A distinct co-expressed sulfurtransferase extends the physiological role of mercaptopropionate dioxygenase in Pseudomonas aeruginosa PAO1.

Oxidation and assimilation of persulfides in bacteria is often catalyzed by a persulfide dioxygenase and sulfurtransferase in consecutive reactions. Enzymes responsible for the oxidation of persulfides have not been clearly defined in Pseudomonas aeruginosa PAO1. The characterized mercaptopropionate dioxygenase (MDO) in P.

Current understanding of enzyme structure and function in bacterial two-component flavin-dependent desulfonases: Cleaving C-S bonds of organosulfur compounds.

The inherent structural properties of enzymes are critical in defining catalytic function. Often, studies to evaluate the relationship between structure and function are limited to only one defined structural element. The two-component flavin-dependent desulfonase family of enzymes involved in bacterial sulfur acquisition utilize a comprehensive range of structural features to carry out the desulfonation of organosulfur compounds.

Oligomeric Changes Regulate Flavin Transfer in Two-Component FMN Reductases Involved in Sulfur Metabolism.

The FMN reductases (SsuE and MsuE of the alkanesulfonate monooxygenase systems) supply reduced flavin to their partner monooxygenases for the desulfonation of alkanesulfonates. Flavin reductases that comprise two-component systems must be able to regulate both flavin reduction and transfer. One mechanism to control these distinct processes is through changes in the oligomeric state of the enzymes.

Shorter Alkanesulfonate Carbon Chains Destabilize the Active Site Architecture of SsuD for Desulfonation.

Bacteria have evolved to utilize alternative organosulfur sources when sulfur is limiting. The SsuE/SsuD and MsuE/MsuD enzymes expressed when sulfur sources are restricted, are responsible for providing specific bacteria with sulfur in the form of alkanesulfonates. In this study, we evaluated why two structurally and functionally similar FMNH-dependent monooxygenase enzymes (MsuD and SsuD) are needed for the acquisition of alkanesulfonates in some bacteria.

On 'Tissue sulfhydryl groups' by George L.Ellman.

This commentary features the groundbreaking manuscript published in the 1959 issue of Archives of Biochemistry and Biophysics by George L. Ellman. The studies describe the quantification of thiols in tissues and purified proteins using DTNB (Ellman's Reagent).

The influence of personal attitude on instructor intent to integrate online collaborative activities.

Collaborative activities are a method used in higher education to develop the higher-order skills that students need to succeed in today's workforce. However, instructors have continued to make the integration of online collaborative activities a low priority. The purpose of this study was to explore the influence of personal attitude on instructor intent to integrate collaborative activities.

Bacterial flavoprotein monooxygenase YxeK salvages toxic S-(2-succino)-adducts via oxygenolytic C-S bond cleavage.

Thiol-containing nucleophiles such as cysteine react spontaneously with the citric acid cycle intermediate fumarate to form S-(2-succino)-adducts. In Bacillus subtilis, a salvaging pathway encoded by the yxe operon has recently been identified for the detoxification and exploitation of these compounds as sulfur sources. This route involves acetylation of S-(2-succino)cysteine to N-acetyl-2-succinocysteine, which is presumably converted to oxaloacetate and N-acetylcysteine, before a final deacetylation step affords cysteine.

Substrate-Dependent Mobile Loop Conformational Changes in Alkanesulfonate Monooxygenase from Accelerated Molecular Dynamics.

Substrate-induced conformational changes present in alkanesulfonate monooxygenase (SsuD) are crucial to catalysis and lead to distinct interactions between a dynamic loop region and the active site. Accelerated molecular dynamics (aMD) simulations have been carried out to examine this potential correlation by studying wild-type SsuD and variant enzymes bound with different combinations of reduced flavin (FMNH), C4a-peroxyflavin intermediate (FMNOO), and octanesulfonate (OCS). Three distinct mobile loop conformations were identified: "open", "closed", and "semiclosed".

The 3-His Metal Coordination Site Promotes the Coupling of Oxygen Activation to Cysteine Oxidation in Cysteine Dioxygenase.

Cysteine dioxygenase (CDO) structurally resembles cupin enzymes that use a 3-His/1-Glu coordination scheme. However, the glutamate ligand is substituted with a cysteine (Cys93) residue, which forms a thioether bond with tyrosine (Tyr157) under physiological conditions. The reversion variant, C93E CDO, was generated in order to reestablish the more common 3-His/1-Glu metal ligands of the cupin superfamily.

Investigations of two-component flavin-dependent monooxygenase systems.

Bacterial two-component flavin-dependent monooxygenase systems catalyze the oxidation of diverse metabolic reactions. There are several shared mechanistic features in the two-component monooxygenase systems that differ from canonical monooxygenase enzymes. The flavin reductases catalyze the reductive half-reaction, and the reduced flavin is transferred to the monooxygenase enzyme.

Not as easy as π: An insertional residue does not explain the π-helix gain-of-function in two-component FMN reductases.

The π-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The π-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4-helix. Variants of Tyr118 were generated, and their X-ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π-helix.

Functional Evaluation of the π-Helix in the NAD(P)H:FMN Reductase of the Alkanesulfonate Monooxygenase System.

A subgroup of enzymes in the NAD(P)H:FMN reductase family is comprised of flavin reductases from two-component monooxygenase systems. The diverging structural feature in these FMN reductases is a π-helix centrally located at the tetramer interface that is generated by the insertion of an amino acid in a conserved α4 helix. The Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme.

Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction.

Chaperonin and cochaperonin, represented by E. coli GroEL and GroES, are essential molecular chaperones for protein folding. The double-ring assembly of GroEL is required to function with GroES, and a single-ring GroEL variant GroEL forms a stable complex with GroES, arresting the chaperoning reaction cycle.

Transformation of a Flavin-Free FMN Reductase to a Canonical Flavoprotein through Modification of the π-Helix.

The flavin reductase of the alkanesulfonate monooxygenase system (SsuE) contains a conserved π-helix located at the tetramer interface that originates from the insertion of Tyr118 into helix α4 of SsuE. Although the presence of π-helices provides an evolutionary gain of function, the defined role of these discrete secondary structures remains largely unexplored. The Tyr118 residue that generated the π-helix in SsuE was substituted with Ala to evaluate the functional role of this distinctive structural feature.

Exposing the Alkanesulfonate Monooxygenase Protein-Protein Interaction Sites.

The alkanesulfonate monooxygenase enzymes (SsuE and SsuD) catalyze the desulfonation of diverse alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase enzyme. Previous studies have highlighted the presence of protein-protein interactions between SsuE and SsuD thought to be important in the flavin transfer event, but the putative interaction sites have not been identified.

Shifting redox states of the iron center partitions CDO between crosslink formation or cysteine oxidation.

Cysteine dioxygenase (CDO) is a mononuclear iron-dependent enzyme that catalyzes the oxidation of L-cysteine to L-cysteine sulfinic acid. The mammalian CDO enzymes contain a thioether crosslink between Cys93 and Tyr157, and purified recombinant CDO exists as a mixture of the crosslinked and non crosslinked isoforms. The current study presents a method of expressing homogenously non crosslinked CDO using a cell permeative metal chelator in order to provide a comprehensive investigation of the non crosslinked and crosslinked isoforms.

Crystal structure of Escherichia coli SsuE: defining a general catalytic cycle for FMN reductases of the flavodoxin-like superfamily.

The Escherichia coli sulfur starvation utilization (ssu) operon includes a two-component monooxygenase system consisting of a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase, SsuE, and a monooxygenase, SsuD. SsuE is part of the flavodoxin-like superfamily, and we report here the crystal structures of its apo, FMN-bound, and FMNH2-bound forms at ∼2 Å resolution. In the crystals, SsuE forms a tetramer that is a dimer of dimers similar to those seen for homologous FMN reductases, quinone reductases, and the WrbA family of enzymes.

Exploring the catalytic mechanism of alkanesulfonate monooxygenase using molecular dynamics.

The complex mechanistic properties of alkanesulfonate monooxygenase (SsuD) provide a particular challenge for identifying catalytically relevant amino acids. In response, a joint computational and experimental study was conducted to further elucidate the SsuD mechanism. Extensive unbiased molecular dynamics (MD) simulations were performed for six SsuD systems: (1) substrate-free, (2) bound with FMNH2, (3) bound with a C4a-peroxyflavin intermediate (FMNOO(-)), (4) bound with octanesulfonate (OCS), (5) co-bound with FMNH2 and OCS, and (6) co-bound with FMNOO(-) and OCS.

Steady-state kinetic isotope effects support a complex role of Arg226 in the proposed desulfonation mechanism of alkanesulfonate monooxygenase.

The alkanesulfonate monooxygenase system catalyzes the desulfonation of alkanesulfonates through proposed acid-base mechanistic steps that involves the abstraction of a proton from the alkane peroxyflavin intermediate and protonation of the FMN-O(-) intermediate. Both solvent and kinetic isotope studies were performed to define the proton transfer steps involved in the SsuD reaction. Substitution of the protium at the C1 position of octanesulfonate with deuterium resulted in an observed primary isotope effect of 3.

Pulsed EPR study of amino acid and tetrahydropterin binding in a tyrosine hydroxylase nitric oxide complex: evidence for substrate rearrangements in the formation of the oxygen-reactive complex.

Tyrosine hydroxylase is a nonheme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin, and molecular oxygen. We have used nitric oxide as an O₂ surrogate to poise Fe(II) at the catalytic site in an S = 3/2, {FeNO}⁷ form amenable to EPR spectroscopy.

Identification of critical steps governing the two-component alkanesulfonate monooxygenase catalytic mechanism.

The alkanesulfonate monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of a carbon-sulfur bond from sulfonated substrates. A mechanism involving acid-base catalysis has been proposed for the desulfonation mechanism by SsuD. In the proposed mechanism, base catalysis is involved in abstracting a proton from the alkane peroxyflavin intermediate, while acid catalysis is needed for the protonation of the FMNO(-) intermediate.

Deletional studies to investigate the functional role of a dynamic loop region of alkanesulfonate monooxygenase.

Several bacterial organisms rely on the two-component alkanesulfonate monooxygenase system for the acquisition of organosulfonate compounds when inorganic sulfur is limiting in the environment. This system is comprised of an FMN reductase (SsuE) that supplies reduced flavin to the alkanesulfonate monooxygenase (SsuD). Desulfonation of alkanesulfonates by SsuD is catalyzed through the activation of dioxygen by reduced flavin.

Addition of an external electron donor to in vitro assays of cysteine dioxygenase precludes the need for exogenous iron.

Cysteine dioxygenase (CDO) utilizes a 3-His facial triad for coordination of its metal center. Recombinant CDO present in cellular lysate exists primarily in the ferrous form and exhibits significant catalytic activity. Removal of CDO from the reducing cellular environment during purification results in the loss of bound iron and oxidation of greater than 99% of the remaining metal centers.

Mechanism for sulfur acquisition by the alkanesulfonate monooxygenase system.

The bacterial alkanesulfonate monooxygenase system is involved in the acquisition of sulfur from organosulfonated compounds during limiting sulfur conditions. The reaction relies on an FMN reductase to supply reduced flavin to the monooxygenase enzyme. The reaction catalyzed by the alkanesulfonate monooxygenase enzyme involves the carbon-sulfur bond cleavage of a wide range of organosulfonated compounds.

Functional role of a conserved arginine residue located on a mobile loop of alkanesulfonate monooxygenase.

The structure of the flavin-dependent alkanesulfonate monooxygenase (SsuD) exists as a TIM-barrel structure with an insertion region located over the active site that contains a conserved arginine (Arg297) residue present in all SsuD homologues. Substitution of Arg297 with alanine (R297A SsuD) or lysine (R297K SsuD) was performed to determine the functional role of this conserved residue in SsuD catalysis. While the more conservative R297K SsuD possessed a lower k(cat)/K(m) value (0.